Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.     

At the edge and on the top: molecular identification and ecology of Daphnia dentifera and D. longispina in high-altitude Asian lakes

The occurrence of members of the highly diverse Daphnia longispina complex in Southern and Central Asian high-mountain lakes has been recognized for more than a century. Until now, however, no molecular data have been available for these populations inhabiting the “Roof of the World.” Here, we present the first identification for D. gr. longispina from that region based on a molecular phylogeny. Our findings show that alpine lakes in the Pamir and Himalaya mountains host populations of widespread species of the complex, for which these are the highest known localities. A spineless morph from the Himalaya region, previously labeled as D. longispina var. aspina, was clustering tightly with D. dentifera, while a population from the Pamir mountain range was grouped with D. longispina. In addition, we analyzed ecological data available for lakes in the Khumbu region (Himalaya) to investigate ecological preferences of non-pigmented D. gr. longispina. The identified factors can at least partly be related to avoidance of high UV conditions by this species. We conclude that the widespread species D. dentifera and D. longispina also colonized the Asian high-mountain lakes, and identify the need for further research to trace the possible effect of rapid environmental changes in this region on the diversity and ecology of high-altitude Daphnia populations.     

Anti-influenza active and low toxic N-phenyl-substituted β-amidoamidines structurally related to natural antibiotic amidinomycin

A set of racemic N-phenyl-substituted β-amidoamidines hydrochlorides 4, which are structurally related to natural antiviral agent amidinomycin (1), was synthesized in four steps starting from methacryloyl anilide (5). In the final step of the synthetic route, an uncommon monoacylation of β-aminoamidine 8 at the less reactive β-phenylamino-group took place. To rationalize this result, a mechanism which involves initial acylation at the more active amidine-function followed by intramolecular acyl-group transfer to β-phenylamino-group was suggested. All three β-amidoamidines 4d–f bearing long linear aliphatic chain (from n-C₈H₁₇ to n-C₁₂H₂₅) revealed significant in vitro activity against influenza A virus (H3N2) and modest cytotoxicity. The in vitro antiviral potency of 4d,e is 6–20 times greater than that of commercial rimantadine with lower EC₅₀ values and higher therapeutic index. The non-toxic in vivo compounds 4d–f showed a beneficial protective effect in influenza A (H3N2) infected mice.     

Magnesium deficiency results in an increased formation of osteoclasts

Magnesium (Mg²⁺) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg²⁺ deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg²⁺ restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg²⁺ (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg²⁺ deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg²⁺-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP⁺) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg²⁺ deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg²⁺ deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg²⁺ deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg²⁺. In conclusion, Mg²⁺ deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg²⁺ deficient patients.     

Novel thiol- and thioether-containing amino acids: cystathionine and homocysteine families

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation reactions of sulfur nucleophiles, such as the ester of l-cysteine and potassium thioacetate.     

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic–clonic seizures and reduced the time spent in the generalized tonic–clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na⁺, K⁺-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.